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Introduction
Phage display has been proven as a highly effective
approach for designing cytokine variants and developing small
molecular drugs[1]. As evidenced by numerous studies within
the past decade, the extracellular domain of cytokine
receptors can be mapped by screening peptide libraries, and small
mimetic peptides corresponding to the active binding area
of their cognate cytokines can be consequently identified
and developed as macromolecular drug
substitutes[2]. Wrighton et al isolated a 14 amino acid peptide targeted to
the erythropoietin (EPO) cytokine receptor. This small
peptide specifically bound the EPO receptor and activated the
appropriate signaling pathways, while also retaining
biological activity[3]. Likewise, Cwirla
et al identified a 14 amino acid peptide from recombinant peptide libraries. This peptide
displayed high affinity to the human thrombopoietin (TPO)
receptor and stimulated the proliferation of a TPO-responsive
Ba/F3 cell line[4]. Another group used human CD81
(hCD81)-expressing murine NIH/3T3 cells to select a hCD81-binding
peptide from a phage-display nonapeptide library. The
motif-containing phages were able to competitively inhibit the
binding of hepatitis C virus enveloping glycoprotein 2 to
native hCD81-expressing MOLT-4 cells[5]. In the same manner,
many mimetic peptides of various cytokines have been
harvested, including melanocytes-stimulating hormone
(MC)[6], estrogen[7], interleukins (IL), and
TNF[8,9].
Interferon (IFN) is currently the human protein most
widely used as a therapeutic agent, having been approved
to treat various types of viral diseases. However, its efficient
treatment is often hampered by various side-effects, such as
neutralizing antibodies and cell
resistance[10,11]. In overcoming these difficulties, the mimetic or synthetic peptide
approach is very attractive. For instance, Sato
et al described the successful mimicry of
IFN-β by a peptide isolated from phage-display screening using a neutralizing
anti-IFN-β monoclonal antibody. Despite the lack of similarity to the
primary sequence of IFN-β, this 15-mer peptide retained the
ability to bind the type I IFN receptor, as well as the antiviral
activity of IFN-β[12]. Based on a similar strategy, Hu
et al selected 2 peptides using a neutralizing antibody from a
phage-display hexapeptide library; one of these peptides
(WLDPRH) exhibited structural homology with loop AB
(29_35), the binding domain of the type I IFN receptor. Although
it exhibited no activity alone, this peptide was evidently able
to cooperatively improve the antiviral activity of
IFN-α[13]. In yet another example, a synthetic peptide corresponding
to residues 95_133 of murine IFN-γ was shown to possess
antiviral activity[14].
In fact, it should be a straightforward strategy to obtain
agonist peptides that bind to and stimulate the IFN receptor
(IFNAR) by screening a phage-display library with the IFN
receptor. However, there are some technical difficulties in
the purification and maintenance of the native structure when
working with membrane receptors. Previous studies have
shown that screening with a soluble form of the type I IFN
receptor failed to identify any peptide
ligands[12]. Therefore, it appears better to use whole native or transfected cells to
select ligands[15,16]. Whole cells usually produce receptors
that maintain the native conformation with normal
post-translational modification, so ligands can be selected even
without detailed information on the nature of the
receptor[5]. However, there have been few reports of IFN mimetic
peptides possessing antiviral activity being discovered by
direct cell-based selection. In a previous
study[17], we performed 4 rounds of biopanning against intact native WISH cells
expressing IFNα-2b receptors, followed by 3 rounds of
selection with polyclonal anti-IFNα-2b antibodies. This
approach resulted in the enrichment of phage clones binding
both to IFNα-2b receptors and anti-IFNα-2b antibodies.
Unfortunately, almost all of the phage clones randomly
picked out from the last round of selection were antagonist
peptides of IFNα-2b. In the current study, we developed a
functional selection strategy to obtain antiviral mimetic
peptides of IFNα-2b in the hopes of designing IFN mimetic
peptide agonists that bind with higher affinity to the receptor.
Materials and methods
Reagents IFNα-2b standard
(1.0×1011 IU/g IFN protein) was kindly provided by Tianjin Hualida Bioengineering
(Tianjin, China). Cell culture media and reagents were
obtained from Gibco BRL Life Technologies (Gaithersburg, MD,
USA). The phage-display heptapeptide (PhD-7) library kit
(1.5×1016 plaque-forming units [pfu]/L) was purchased from
New England Biolabs (Beverly, MA, USA). Wild-type M13
phage and the horseradish peroxidase (HRP)-conjugated
anti-M13 phage antibody were purchased from Pharmacia
Biotech (Uppsala, Sweden). The fusion protein
GFP/IFNα-2b was prepared as outlined in our previous
studies[17]. Other chemicals used in this study were of analytical grade and
were commercially available.
Cell culture WISH cells and vesicular stomatitis virus
(VSV) were kindly provided by Hualida Bioengineering. The
WISH cells, which endogenously express type I IFN
receptors on the cell surface, were cultured at 37 °C with 5%
CO2 in RPMI-1640 medium supplemented with 10%
(v/v) heat-inactivated fetal bovine serum,
1×105 U/L penicillin, and 0.1 g/L streptomycin.
Functional selection The primary PhD-7 library, which
had been previously subjected to 4 rounds of biopanning
against WISH cells as described[18], was used for the
functional selection. The selection procedure was based on
combining the PhD-7 library kit standard procedure with some
modifications for functional screening for the protection of
human amnion WISH cells against VSV-induced cytopathic
effects[18]. WISH cells
(2.0×104) were seeded into 96-well
culture plates and incubated at 37 °C for 3 h.
Approximately 1.0×1011 pfu phage were transferred to the 96-well culture
plates and incubated at 37 °C for 24 h. The cells were then
challenged with VSV and incubated at 37 °C for an additional
24 h. After washing 10 times with RPMI-1640 medium and 5
times with phosphate-buffered saline (PBS; 0.02 mol/L
sodium phosphate buffer, pH 7.4, and 0.15 mol/L NaCl); the
remaining phages that had bound to surviving cells were
eluted for 30 min with 0.01 g/L IFNα-2b. The eluted phages
were replicated by infecting Escherichia
coli ER2738 cells. The amplified phage particles were purified using
polyethylene glycol and then used for subsequent rounds of selection.
Phage ELISA Phage ELISA was performed according to
our previous report[17]. Briefly, approximately
1.0×1010 pfu-amplified phages (or mixed with 0.01 g/L
IFNα-2b) were incubated with WISH cells
(2.0×105 cells/well) fixed onto 96-well
culture plates with glutaraldehyde. After extensive washing
with PBST (PBS containing 0.1% Tween 20), the bound
phages were detected with the HRP-conjugated anti-M13
phage antibody. Wild-type M13 phage was used as a negative
control.
Peptide sequencing and synthesis The single-stranded
DNA (ssDNA) was prepared from identified phage clones as
described in the PhD-7 library kit guidelines and sequenced
by the Shanghai Sangon Company (Shanghai, China).
Corresponding amino acid sequences were deduced from DNA
sequences; 2 peptides corresponding to positive clones T3
and T9 and their corresponding IFN sequences were
synthesized chemically by GL Biochem (Shanghai, China) and
designated as IR-7 (clone T3, IRPDTPR), VR-7
(IFN143-149, VRAEIMR), KP-7 (clone T9, KNVHPPP) and KG-7
(IFN31-37, KDRHDFG). Two antagonist peptides obtained from
previous studies[17], SP-7(SLSPGLP) and FY-7(FSAPVRY), were
also used as the controls. At the same time, the 4 mutant
peptides of T3 and T9 were synthesized and used in antiviral
activity assay, and designated as IR-7A (IGPDTPR),
IR-7B (IRPDTPG), KP-7A (GNVHPPP) and KP-7A (KNVGPPP). The
key residues, Arg144 or Arg149 of IR-7 and Lys31 or His34 of
IR-7, were replaced with Gly, respectively.
Synthetic peptide competition assay
Exponentially-growing WISH cells were fixed on culture plates using
glutaraldehyde (2.0×105 cells/well). After blocking with PBS
(containing 1% bovine serum albumin and 0.1%
NaN3) for 1 h at room temperature, 1 µg
GFP/IFNα-2b was added to each well and incubated at 37 °C for 2 h. After washing
5 times with PBST, different amounts of synthetic peptides were
added and incubated at 37 °C for 1 h with gentle shaking.
The wells were then washed with PBST again and observed
with an inverted fluorescence microscope. The intensity of
fluorescence was analyzed with Image Pro Plus 5.1 and the
IC50 values were calculated with GraphPad Prism 4
software[19].
Antiviral activity assay The antiviral activity of IFN and
synthetic peptides was determined in vitro by the
protection of human amnion WISH cells against VSV-induced
cytopathic effects, as described for the traditional
method[18]. In brief,
2.0×104 WISH cells were seeded into each well of
96-well plates and incubated with samples for 24 h at 37 °C.
After incubation, the cells were challenged with VSV and the
plates were incubated at 37 °C for 24 h. Virus-induced
cytopathic effects were assayed by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method[20].
Results
Specific enrichment of positive phages with antiviral
activity In order to efficiently enrich IFNAR-binding phages
with functional antiviral activity, we chose a phage-display
library featuring specific binding to IFNAR as the primary
library for further functional selection. The functional
enrichment was determined by the antiviral activity after each
round of selection. In 3 rounds of functional selection, the
antiviral activity increased markedly after each round
compared to the primary library (Figure 1), indicating enrichment
for positive phages with antiviral activity.
Identification of the positive phages Clones (1012) were
picked out from the sample after the third round of
functional selection and the antiviral activity was examined. Only
16 clones (approximately 1.6%) showed antiviral activity
(Figure 2). Further phage ELISA testing demonstrated that
12 out of the 16 positive clones (T1_9, 11, 12, 14, 15, 17, 18,
and 20) could bind to WISH cells, and 10 out of these 12
binding clones (T3_7, 9, 12, 15, 17, and 18) could be
specifically competed off by 1 µg IFNα-2b (Figure 2). These results
confirmed the success of the functional selection scheme in
enriching IFNAR-binding phages with antiviral activity.
Analyses of exogenous sequences of positive-phage
clones The ssDNA prepared from the 10 positive clones
were sequenced, and the amino acid sequences of the
mimetic peptides were then deduced from the DNA sequences.
A homologous analysis of amino acid sequences revealed
that the selected clones were corresponding to 7 domains
defined by residues of IFNα-2b (T9: AB loop 31_37; T18: BC
loop 68_74; T4: C helix 93_99; T6, T17: CD loop 105_112;
T12: D helix 115_121; T5: DE loop 132_138 and T3, T7, T15: E
helix 143_161; Table 1). It has been proposed that the AB
loop (residues 26_35) and E helix (residues 144_153) mediate
the interaction with receptors[13]. By comparing the
homology with the amino acid sequence of the known functional
domain in IFNα-2b, 2 peptides represented by the positive
clones T3 and T9 and aligned with the IFNAR2-binding
domains (AB loop and E helix), their mutant peptides, and their
corresponding native IFN sequences were synthesized and
designated IR-7, KP-7, IR-7A, IR-7B, KP-7A, KP-7B, VR-7,
and KG-7, respectively.
Competition reactivity of mimetic peptides to IFNAR
The abilities of 2 mimetic peptides, IR-7 and KP-7, to inhibit the
binding of GFP/IFNα-2b to IFNAR were determined by a
competitive assay. As shown in Figure 3, IR-7 and KP-7
reduced fluorescence-bound GFP/IFNα-2b in a
dose-dependent manner as well as 2 antagonist peptides, SP-7 and FY-7,
reported previously[17]. The
IC50 values of IR-7 and KP-7 were 16.8 and 11.9 nmol, respectively, slightly higher than SP-7
(11.2 nmol) and FY-7 (4.03 nmol). These results confirmed
that 2 mimetic peptides did effectively mimic IFNα-2b and
inhibited the binding of GFP/IFNα-2b to IFNAR.
Antiviral activity of mimetic peptides The antiviral
activity of the mimetic peptides was determined by the MTT
method. When the peptide was added with an amount
between 1.5 and 6.0 nmol, both mimetic peptides (IR-7 and
KP-7) showed significant antiviral activity in a dose-dependent
manner compared to their corresponding IFN sequences
peptides (VR-7 and KG-7) and the antagonist control
peptides (SP-7 and FY-7; Figure 4), which suggested that the
secondary structure of peptides was very important in
antiviral activity. At the same time, these experiments indicated
that the antiviral activity of KP-7 was slightly stronger than
that of IR-7. Furthermore, the antiviral activity of a KP-7
and IR-7 mixture was even stronger still than that of KP-7 or IR-7
alone at the same dosage, indicating that the 2 domains, the
AB loop (residues 31_37) and E helix (residues 143_149), in
IFNα-2b can inhibit viral infection cooperatively.
Comparing IR-7 or KP-7 with its mutant peptides, the antiviral
activity of original mimetic peptides was obviously stronger than
its mutant peptides (Figure 5), and this result suggested that
the key residue was indispensable.
Discussion
Type I IFN is a family of homologous cytokines that
potently elicits an antiviral and antiproliferative state in cells.
All human type I IFN (IFN-α, -β, -τ, and -ω) bind to a cell
surface receptor consisting of 2 transmembrane subunits,
IFNAR1 and IFNAR2, that associate on binding. The
binding of IFN to its receptor triggers a cascade of events,
activating a number of proteins that inhibit viral replication and
cell growth[21].
In previous attempts to develop antagonist peptides to
IFNα-2b, we performed biopanning against WISH cells and
polyclonal anti-IFN antibodies. The method provided a
final-phage display library in which the phage clones could
bind both to IFN receptors and anti-IFN
antibodies[17]. However, many of these clones might not activate the
receptors to stimulate the IFN signaling pathway. Building on
these tactics, we developed a third strategy called
"functional biopanning selection" in order to obtain mimetic
peptides of IFNα-2b with antiviral activity. In this functional
selection procedure, VSV was used to infect WISH cells that
had been treated with the phage-display library. After
incubation, most of the infected WISH cells had died and
could be washed away together with phage clones without
antiviral activity. However, on the surface of the surviving
cells, we anticipated that there might be combined phage
clones possessing protective and antiviral activity, and that
these candidates could be competitively eluted by
IFNα-2b. Our results confirmed that positive phages with antiviral
activity had indeed been enriched after 3 rounds of
functional panning selection (Figure 1).
Although Figure 1 shows that the survival of WISH cells
in the third round was approximately 60% compared with the
positive control, the positive hit rate was only 1.6%. One
explanation for this phenomenon is that there are plenty of
IFN receptors on the surface of WISH cells, but the
activation of only a few can result in antiviral
activities[22]. Therefore, most of phage clones synchronously bound to IFN
receptors on surviving WISH cells during the functional selection,
but some binding was irrelative with antiviral activity. Among
the 12 positive phage clones selected on WISH cells, 10
clones could bind to IFNAR specifically (T3_7, 9, 12, 15, 17,
and 18), so the antiviral activities of these clones should be
related to the IFN signaling pathway.
It has been recently determined by X-ray
crystallography that the IFNα-2b molecule is composed of
5 α-helices (A_E) linked by 1 long connection (AB loop) and 3 short
segments (the BC, CD, and DE loops)[23]. In this study, the
sequences of 10 positive clones were corresponding to
7 domains defined by residues of IFNα-2b (T9: AB loop
31_37; T18: BC loop 68_74; T4: C helix 93_99; T6, T17: CD loop
105_112; T12: D helix 115_121; T5: DE loop 132_138 and T3,
T7, T15: E helix 143_161; Table 1). The A helix (residues
12_15), the AB loop (residues 26_35), and the E helix (residues
144_153) of IFN-α binds IFNAR2, triggering the induction
of antiviral activity[13,24]. Therefore, it is possible that the
peptides of T9 are structural mimics of conformational
epitopes of the IFN AB loop, and those of T3, T7, and T15
might be E helix mimics.
Mitsui et al and Uzé et al's studies revealed that if the
amino acid sites of Leu30, Lys31, Arg33, His34, Phe36, Arg120,
Lys121, Tyr122,Gln124, Tyr129, Lys131, and Glu132 on
IFNα are mutated, the bioactivity is observably
reduced[25,26]. This indicates that these sites are important for IFN-introduced
antiviral activity; however, Lys31 and His34 of T9 lie within
this range. In Jacob's research, 4 conservative amino acids,
Arg144, Ala145, Met148, and Arg149 in the E helix were
identified as "hot amino acids" key points for the interaction
between IFN and its receptor[21,27]. Clone T3 includes Arg144
and Arg149. In addition, similarity analysis indicated that
Glu146 in IFN and Asp146 of T3 were equivalent, since they
are both acidic amino acids. We thus selected clones T9 and
T3 for peptide synthesis, designated as KP-7 and IR-7. These
2 peptides were not only capable of specific binding to the
IFN receptor (Figure 3), but also provided antiviral activity
in a dose-dependent manner (Figure 4). Furthermore, we
observed a cooperative effect on antiviral activity when both
peptides were added simultaneously. To confirm the
function of key residues from mimetic peptides, we synthesized 4
mutant peptides at key residue (Lys31 and His34 of T9; Arg144
and Arg149 of T3) and performed an antiviral activity assay.
The results indicated again that the key points were
indispensable in the IFN antiviral process.
In this study, we completed only preliminary studies for
T3 and T9, although other positive clones also had some
antiviral activity. For example, T4 (93_99) partially overlaps
with the C helix, which is involved in the binding of IFN to
IFNAR1[28]. In addition, Ser68 of T18, Arg120 of T12, and
Lys133 of T5 are known to be important amino acids for IFN
bioactivity. Therefore, these clones also hold interest for
further studies.
Using cell selection and functional biopanning,
2 IFNα-2b agonist peptides with antiviral activity, KP-7 and IR-7,
were successfully selected from a random phage-display
library. The results of this novel functional biopanning
technique suggest that IFNAR-induced antiviral activity may
require multiple binding domains, an idea that may prove
helpful in further elucidating the molecular mechanism of
IFN receptor binding. Moreover, this functional biopanning
technique should be useful in the development of mimetic
peptide drugs of human IFN and other cytokines.
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